Influence of antibacterial surface treatment on dental implants on cell viability: A systematic review

There is no consensus in the literature about the best non-cytotoxic antibacterial surface treatment for dental implants. Critically evaluate the existing literature and answer the question: “which surface treatment for dental implants made of titanium and its alloys has the greatest non-cytotoxic antibacterial activity for osteoblastic cells?” This systematic review was registered in the Open Science Framework (osf.io/8fq6p) and followed the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols. The search strategy was applied to four databases. Articles were selected that evaluated in both studies the properties of 1) antibacterial activity and 2) cytotoxicity on osteoblastic cells of titanium and their alloy dental implants when treated superficially. Systematic reviews, book chapters, observational studies, case reports, articles that studied non-dental implants, and articles that evaluated only the development of surface treatment were excluded. The Joanna Briggs Institute, a quasi-experimental study assessment tool, was adapted to assess the risk of bias. The search strategy found 1178 articles in the databases after removing duplicates in EndNote Web, resulting in 1011 articles to be evaluated by title and abstract, of which 21 were selected for full reading, of which 12 were included by eligibility criteria, and nine were excluded. Quantitative synthesis could not be performed due to the heterogeneity of the data (surface treatment, antibacterial assay, bacteria strain, cell viability assay, and cell type). Risk of bias assessment showed that ten studies were classified as low risk and two studies as moderate risk. The evaluated literature allowed us to conclude that: 1) The literature surveyed did not allow answering the question due to the heterogeneity of the studies; 2) Ten of the 12 studies evaluated presented surface treatments with non-cytotoxic antibacterial activity; 3) Adding nanomaterials, QPEI, BG, and CS, reduce the chances of bacterial resistance by controlling their adhesion by electrical forces.


Introduction
The development of pro-osteogenic and antibacterial bioactive surfaces is promising for success since implant-supported oral rehabilitations are susceptible to challenging oral and systemic patient conditions, which can hinder the integration of the biomaterial into bone tissue [1,2]. Lindhe e Meyle [3] reported that the risk of peri-implant mucositis is around 80% and peri-implantitis 28-56% after implant surgeries. Thus, the microbial challenge facing the surgery to install dental implants leads to proposals to understand the microbiological, bio-physical-chemical, and structural factors involved in the implant/bone interaction to prevent peri-implant infections and their deleterious effects on treatment success are the focus of study [4][5][6][7][8].
Titanium alloys (Ti) are the materials of choice for dental implants due to their biocompatibility, excellent mechanical and physical-chemical properties, and chemical stability [. However, the most widely used alloy, Ti-6Al-4V, is the subject of discussions, as its elements Al and V are considered neurotoxic [7,[16][17][18][19][20][21][22]. Therefore, beta titanium alloys have become promising in recent years, as they incorporate non-cytotoxic chemical elements such as molybdenum (Mb), tantalum (Ta), zirconium (Zr) and niobium (Nb). As its modulus of elasticity is closer to the bone, adaptive proximal bone remodeling (stress shielding) can be avoided, as it allows for an adequate distribution of the forces to which the implant is subjected [7,16,17,[23][24][25][26].
One of the main causes of implant failure is a peri-implant infection caused by specific microbiota, which is associated with periodontitis. Streptococcus spp. is the pioneer in the colonization of the surgical site and peri-implantitis microbiota and includes Fusobacterium spp., Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Campylobacter rectus, Capnocytophaga rectus, Capnocytophaga spp. and Porphyromonas gingivalis [10,27]; associated with high rates of patient dissatisfaction and cost of rehabilitation treatment. In addition, peri-implant infection is more predisposed in smokers and patients with chronic periodontal disease [9,10,13,14,[27][28][29].
The literature presents surface treatments classified as antibacterial by adding antibacterial ions, drugs, and nanomaterials, and non-antibacterial treatments can potentially reduce bacterial adhesion by reducing the favorable area for bacterial colonization [7,8,27,[30][31][32][33][34][35][36][37][38]]. An et al. [39] demonstrated that the anodizing treatment by increasing the diameter of the TiO2 nanotube reduced bacterial activity. The Kreve and Reis 2021 [40] review infers that the regulation of the electrostatic condition of the surface is a promising treatment for regulating bacterial adhesion on titanium surfaces through long-range forces that dictate attraction or repulsion. Treatments without the addition of antibacterial have the advantage of reducing the risk of bacterial resistance by regulating adhesion through physicochemical and electrostatic changes.
Antibacterial surface treatments include the addition of antibacterial ions such as silver (Ag), copper (Cu), and zinc (Zn) that act by disrupting the bacterial cell wall and inhibiting the activity of respiratory enzymes and DNA replication [41][42][43][44][45][46][47][48]. The addition of antibiotics to synthetic poly (adipic anhydride) ((C6H8O3)n) (PADA) and poly(lactic acid-co-glycolic acid) (PLGA) and natural chitosan (CS) polymers allows direct delivery of the drug to the site desired in lower doses if administered orally. However, it is a complex technique because it involves more than one step to be carried out and presents challenges such as determining the concentration of antibiotic that is antibacterial and non-cytotoxic [7,33].
In addition to the surface mentioned above treatments, the addition of antibacterial nanomaterials has shown promise. Thus, bioactive glass (BG), in addition to being known for its osteoinductive capacity, also has antibacterial activity by modulating the rate of ion release, which influences the pH and osmolarity by changing the physiological conditions of the implantation bed [35,49]. The incorporation of polyethyleneimine quaternary ammonium nanoparticles (QPEI) promotes antibacterial activity by electrostatic interaction with bacterial cells [31]. The synthesis of chimeric peptides and their application on titanium surfaces demonstrates antibacterial activity also by electrostatic interaction and by disruption of the bacterial cell membrane and inhibition of RNA replication [32].
Many superficial treatments are available in the literature, with different mechanisms of action and proposals. However, there is no consensus in the literature about the best non-cytotoxic antibacterial surface treatment for titanium dental implants and their alloys. Therefore, the objective of this systematic review was to critically evaluate the existing literature and answer the question: "which surface treatment for dental implants made of titanium and its alloys has the greatest non-cytotoxic antibacterial activity for osteoblastic cells?"

Material and methods
The systematic review was registered in Open Science Framework (osf.io/5jenw) and followed the Preferred Reporting Items for Systematic Review and Meta-Analyses Protocols [50,51]. The PICOS was structured according to the focus question, "which surface treatment for dental implants made of titanium and its alloys has the greatest non-cytotoxic antibacterial activity for osteoblastic cells?" being: Population = titanium and their alloys surfaces for dental implants; Intervention = any surface treatment; Comparison = control group; Outcomes = antibacterial activity and cytotoxicity for osteoblast cells; and Study design = in vitro studies.
Articles were selected to compose this systematic review from in vitro experimental studies that evaluated the antibacterial activity and cytotoxicity for osteoblastic cells of surface treatments applied on titanium surfaces and their alloys for dental implants. In Table 1 Eligibility criteria.

Inclusion Exclusion
Articles that evaluated in the same study both properties 1) antibacterial activity and 2) cytotoxicity on osteoblastic cells of titanium and their alloys dental implants when treated superficially.
addition, were excluded 1) Did not study dental implants, 2) Did not evaluate cell viability on osteoblast cells, 3) Did not study titanium and their alloys, 4) Did not evaluate the antibacterial activity of surface treatment, 5) Full-text article not available. Also, the eligibility criteria as described in Table 1.
The search strategy was applied in the databases Embase, PubMed, Science Direct, and Scopus on November 17th, 2022, without time and language restrictions (Appendix 1). The citation manager was inserted in the EndNote Web to save and remove duplicates after the citation manager was inserted in the Rayyan web app to analyze the titles and abstracts in the first phase.
The selection process was realized in two phases. In the first phase, the reviereviewer's R.LO.R and J.D.C.T assessment independently the titles and abstracts to find articles that meet eligibility criteria. In the second phase, the articles selected in the first phase were read in full to select articles for this review. The coordinator, A.C.R., solved the doubts in the consensus meeting.
The reviewers independently tabulated data in a Microsoft Excel spreadsheet according to Author, year; Population (alloy, groups); Intervention (surface treatment); Comparison (control); Outcome for antibacterial activity (assay, bacteria, and result); the outcome for cell viability (assay, cell, and result) and, Conclusion were detailed in Tables 2 and 3.
The risk of bias was assessed independently by R.L.O.R and J.D.C.T through the adapted quasi-experimental studies appraisal tool by The Joanna Briggs Institute as previously performed by Gama et al., 2020 [52]. The classification of risk of bias was realized in accordance with answers to the questions. When answered "yes" for all the questions, the risk of bias was low (high methodological quality); when answered "yes" for 6 or 7 of the questions moderate risk of bias (moderate methodological quality), and five or less of the questions high risk of bias (low methodological quality).

Selection process
The search strategy found 1178 articles in the databases. After removing duplicates in EndNote Web, it resulted in 1011 articles to be evaluated by title and abstract, of which 21 were selected for reading in full, of these 12 [7,8,27,[30][31][32][33][34][35][36][37][38] were included in this systematic review because they met the eligibility criteria and nine were excluded (Appendix 2). It is noteworthy that no article was included in the list of references and the gray literature (Fig. 1).

Meta-analysis
The quantitative synthesis could not be carried out due to the heterogeneity of the studies regarding the evaluated titanium alloy, surface treatment, antibacterial activity assay, bacteria, cell viability assay, and osteoblastic cell type.

Risk of bios
The risk of bias assessment showed that ten studies were classified as having a low risk of bias and two studies as having a moderate risk of bias. The study by Zboun et al., 2022 [37] demonstrated increased levels of risk due to the lack of a control group, and Pistilli et al., 2018 [36] for not including similar surface treatments for comparisons (Figs. 2 and 3).

Discussion
The inflammatory response to the pathogenic microbiota caused by peri-implant infections is associated with rates of 32% for the failure of dental implants and 73.2% for lack of osseointegration [1,2]. However, most surface treatments with antibacterial purposes performed on dental implants in order to remedy this gap have cytotoxic effects [31,53]. Therefore, to understand the antimicrobial mechanisms of surface treatments of titanium dental implants and their alloys and their effects both on bacterial activity and on cell The studies that met the eligibility criteria were heterogeneous in relation to the applied surface treatment, titanium alloy, type of cell evaluated, and bacterial strains, which made it impossible to answer the research question and carry out the quantitative metaanalysis. Therefore, a thorough qualitative systematic review was performed to provide the best synthesis of the available literature [7,8,27,[30][31][32][33][34][35][36][37][38].
Surface treatment with bioactive glass has been the subject of research in recent years and has become promising due to the indirect antibacterial action promoted by its chemical composition when interacting with the titanium dioxide layer of the implant [6,35,49]. These inferences are supported by Mokhtari et al., 2018 [35], who compared the application of the BG coating on different substrates (Ti, TNT/QS, and Ti/QS) and observed better rates of antibacterial activity in the TNT-BG group, attributed to the mechanical interaction between TNTs and BG providing a greater amount of QS on the surface. Furthermore, the increase in roughness and hydrophilicity provided by BG allowed a greater proliferation of osteoblastic cells.
Sun et al., 2019 [31] proposed the use of an antibacterial polymer, QPEI, and demonstrated that it presents good osteoblastic cell viability only when associated with ALEN, a drug that is biocompatible with osteoblasts and has high affinity with bone minerals. The antibacterial activity of QPI is attributed to bacterial cell membrane disruption. In addition, it has a low affinity for bacterial adhesion, attributed to the presence of hydroxyl groups in the formation process of this coating with broad-spectrum antibacterial and antifouling properties [54].
The studies of Mokhtari et al., 2018 [35] and Sun et al., 2019 [31] demonstrate that the chemical composition of nanomaterials, BG and QPEI, present molecular arrangements that promote low electrostatic affinity for bacterial cells and high affinity for osteoblastic cells. Thus, promising materials for the development of pro-osteoblastic antibacterial titanium implants.
Innovative local drug delivery systems for dental implants have been proposed to avoid side effects and antibacterial resistance when compared to oral therapy [55]. Kazek-Kęsik et al., 2019 [33] and Leśniak-Ziółkowska et al., 2020 [7] proposed the hybrid coating of polymers PLGA [33] and PADA [7] on Ti-15Mo and Ti-2Ta-3Zr-36Nb surfaces, respectively, treated with PEO associated with antibiotics AMX [7,33], CEF [7] e VAN [7]. The PEO treatment is proposed to be carried out prior to the addition of the polymeric coating, as it promotes an oxidized porous surface that improves the adhesion of the coating. In addition, the literature [7,33] reports that the porous morphology obtained by PEO improves osteoblastic cell viability.
Natural polymers such as CS are also associated with antibiotics and antimicrobials, as demonstrated by Song et al., 2022 [30] and Norowski et al., 2011 [27]. Norowski et al., 2011 [27] demonstrated that the evaluated antimicrobials (tetracycline and chlorhexidine) showed good results regarding their cytotoxicity and antibacterial activity. Tetracycline, when compared to chlorhexidine, presented a prolonged release time due to the electrostatic interaction with CS and did not present a temporary cytotoxic effect.
The non-cytotoxic antibacterial activity of polymeric hybrid coatings with antibiotics is attributed to the slow degradation capacity of the surface layers of polymers, which allows a slow release of antibiotics; this is a favorable characteristic for preventing bacterial adhesion and not inhibiting osteoblastic activity [7,33]. However, the authors of this review infer that it is challenging since more than one processing technique is required and the difficulty of establishing ideal concentrations of the drug in order not to be cytotoxic. Zhou et al., 2019 [38], when preparing patterns of microgrooves with various widths and depths, they attributed the increase in cell viability and antibacterial activity to greater wettability resulting from the increase in depth and width of the microgrooves. Godoy-Gallardo et al., 2016 [8] corroborate this finding by also attributing the increase in antibacterial activity and cell viability to the increased wettability of surfaces treated with the alkaline treatment and the TEPSA silane. In addition, they point out that the presence of the TEPSA silane promoted an increased expression of BMP genes − 2 and RUNX-2, responsible for inducing osteoblastic proliferation.
Zirconium nitride (ZrN) is a nanomaterial that has shown promise for limiting bacterial colonization compared to titanium nitride and other surface treatment approaches such as physical vapor deposition, thermal oxidation, and structuring with laser radiation [56][57][58]. Pistilli et al., 2018 [36] demonstrated that the ZrN coating bioactivated by argon plasma enables higher rates of osteoblastic adhesion and reduction of bacterial activity compared to smooth titanium because its external electronic surface promotes hydrophilicity.    surface treatments that promote hydrophilicity different from pathogenic bacteria can act as anti-adhesives; thus wettability being an important property to consider when developing antibacterial implants.
Bacterial adhesion inhibition can be avoided by the adsorption of chimeric peptides on the surface of titanium and its alloys [32,60]. Geng et al., 2018 [32] proposed hBD3 peptides, which are endogenous human antimicrobial peptides (AMPs) that have broad-spectrum antibacterial activity. Of the peptides tested, TBP-1-GGG-hBD3-3 showed better antibacterial activity than the other two peptides (TBP-1-GGG-hBD3-(1 and 2)), a factor attributed to its greater positive charge, as well as its possible ability to disrupt the bacterial cell membrane and inhibit RNA replication. These findings are promising, as the antibacterial capacity of these peptides can be extended to drug-resistant microbes, in addition to not being cytotoxic to osteoblastic cells, making them a promising approach for antibacterial dental implants.
An ion with excellent antibacterial properties is Ag, as it acts by destroying the bacterial cell wall. Tardelli et al., 2021 [16] inferred that the cytotoxic effect of the chemical element is dependent on 1) dose, 2) exposure time, 3) nanoparticle size, 4) average temperature, and 5) cell type. In this context, López-Ortega et al. [34] 2022 explored the antibacterial effects of incorporating Ag ions (biocidal agent) into Ti nanostructured surfaces by PEO in association with Ca and P ions (bioactive agents) due to their excellent osteoinductive properties. However, the presence of Ag in TNTs showed not only antimicrobial activity but also demonstrated increased levels of expression of osteogenic genes, a fact attributed to the titanium being implanted with Ag in a nanostructured surface. Thus, this study infers that the toxicity of antimicrobial ions such as Ag can be reduced when associated with other biocomposites, corroborating the literature [41][42][43][44]61,62].
On the other hand, Zboun et al., 2022 [37] promoted roughness in titanium surfaces by sandblasting and acid attack (boric acid). Osteoblastic differentiation and antibacterial activity were amplified in this study by the presence of boric acid boron compounds, which offer anti-osteoporotic and anti-inflammatory properties. However, the ideal concentrations of boron have not been clarified and, therefore, motivate future studies that quantify the minimum and maximum concentrations for promoting non-cytotoxic antibacterial activity.
The proposed systematic review, although it cannot answer the question that directed it due to the heterogeneity of the studies, allows inferring those surface treatments that alter the electrostatic condition are by the addition of polymers such as QPEI [31] or antibacterial nanomaterials such as BG [35] and CS [27,30,35] are promising because they allow osteoblastic cell viability and can reduce bacterial resistance rates and side effects when compared to oral antimicrobials and slow local release in polymeric coatings. Thus, electrostatic surface treatments are promising for regulating bacterial adhesion on titanium surfaces through long-range forces, as demonstrated by Kreve and Reis 2021 [40].

Conclusions
1) The literature surveyed in this systematic review, according to PRISMA standards, did not allow answering the question "which surface treatment for dental implants made of titanium and its alloys has the greatest non-cytotoxic antibacterial activity for osteoblastic cells?" due to the heterogeneity of the studies (titanium alloy, surface treatment, antibacterial test, strain, cell, and cell viability test). 2) Of the 12 studies evaluated in this systematic review, ten studies presented surface treatments with non-cytotoxic antibacterial activity. Meanwhile, the addition of calcium phosphate, chitosan, and cefazolin in pulsed current and QPEI in Ti were cytotoxic to osteoblastic cells despite being antibacterial. 3) Of the evaluated surface treatments, those that altered the electrostatic condition of the surface by adding nanomaterials, QPEI, BG, and CS, are considered promising because they reduce the chances of bacterial resistance by controlling their adhesion by electrical forces.

Author contribution statement
Renan Rigotti, Juliana Dias Corpa Tardelli and Andrea Candido Reis: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interest's statement
The authors declare no conflict of interest.